RESEARCH SUMMARY The role that diet plays in brain development will be probed by determining the components of the brain that are directly impacted by nutrition. Human milk is highly glycosylated containing short oligosaccharide chains that are more abundant than proteins. In early nutrition, there is a big burden on diet to accommodate the rapidly developing brain. The brain is rich in sialic acids, which are involved in neuronal outgrowth thereby creating synaptic connectivity through cell-cell interactions that in turn create memory. Human milk oligosaccharides are rich in sialic acids. It has long been proposed that sialic acids in human milk are an important source of sialic acids in the brain. However, the compounds in the brain that incorporate the exogenous sialic acids are not known, although they are generally believed to include proteins and lipids. This research will determine with high specificity the glycoconjugates in the brain that incorporate exogenous monosaccharides, particularly those from human milk oligosaccharides during the normal course of breast- feeding. A method combining advanced separation, mass spectrometry and chemical biology will be developed to determine the glycan structures, the glycoprotein, and the glycolipid involved in the incorporation of glycans from breast milk. Structural heterogeneity will be determined with lipid-specific characterization. Glycans in glycoproteins will be determined with structural heterogeneity at the site-specific level for both N- and O- glycosylation sites. We will develop deep glycomics tools to probe the glycoconjugates as glycoproteins and glycolipids that incorporate components from human milk. Determining the specific protein and lipid glycoconjugates that incorporate milk oligosaccharides would provide the most direct link between nutrition and brain development. 1